In this application we propose to study the phosphoregulatory mechanism and specificity of the MAP kinase ERK2 using single crystal x-ray analysis. Protein kinases are essential molecules in both initiation of signal transduction and in the regulation and integration of cellular processes. The MAP kinase ERK2 has been widely studied as a growth-factor activated, tyrosine phosphorylated enzyme of 41 kDa. This enzyme is activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells, indicating that it is a pleiotropic regulatory enzyme. Of proteins in the kinase superfamily, ERK2 is an especially appropriate target for crystallographic studies. ERK2 has a complex and interesting mechanism of regulation that involves obligate dual phosphorylations, on a single tyrosine and a single threonine residue. It lacks associated regulatory proteins, so that through the study of the single polypeptide the regulatory mechanism can be understood. We have crystallized ERK2 in its inactive unphosphorylated conformation and have refined the structure at 2.3 Angstroms resolution. By comparing the structure of the unphosphorylated enzyme with the active conformation of PKA (cAMP-dependent protein kinase) and by mutational analysis, it has been possible to propose a partial model for the phosphoregulation of ERK2. This model will be tested with crystallographic studies of mutant ERK2 molecules in their dephosphorylated states. Doubly phosphorylated and singly phosphorylated forms have been or will be obtained through the action of bacterially expressed MEK (MAP kinase/ERK kinase). These phosphorylated enzymes will be studied crystallographically to elucidate the structural basis for the complex phosphoregulation of ERK2.